This application seeks to improve basic understanding of the molecular mechanisms that mediate cytoprotective actions of coagulation proteases on cells. Available therapeutic solutions for vascular, thrombotic, and inflammatory diseases are limited and mortality rates remain unacceptably high. Activated protein C (APC) has beneficial effects of in a broad spectrum of injury and disease models where ischemia and inflammation contribute to pathogenesis. The cytoprotective activities of APC are responsible for these beneficial effects of APC in vivo and involve the endothelial protein C receptor (EPCR), protease activated receptor (PAR) 1, and PAR3. The cytoprotective effects of APC on cells contrast with proinflammatory effects of other coagulation proteases (e.g. thrombin) on cells and initiated novel perspectives on how the same receptor (PAR1) can mediate such different opposing effects. The EPCR-dependent non-canonical activation of PAR1 at Arg46 and PAR3 at Arg41 are newly discovered mechanisms of biased PAR signaling. The functional selectivity of thrombin versus APC is thus determined by the proteolytic activation sites in PAR1 and PAR3 that give rise to the new N-terminal tethered-ligands. The immediate goal of this application is to gain novel insights into the molecular mechanisms of non-canonical biased PAR signaling and the extent to which the canonical/non-canonical PAR profile determines the functional selectivity of proteases. Major focus will be on structure-function studies of PAR3 non-canonical signaling mechanisms and of PAR canonical/non- canonical proteolysis profiles of coagulation proteases. Furthermore, several conceptual key aspects of the non-canonical PAR activation and biased signaling paradigm will be tested in vitro and in vivo using engineered EPCR variants that are expected to augment cytoprotective mechanisms. The long-term objectives of this application are to contribute to diagnostic and therapeutic progress for vascular, thrombotic, and inflammatory diseases by advancing knowledge through both basic and translational research. Novel hypotheses will be tested using biochemical and cellular biology methods and validated by studies in mouse models in vivo. The specific aims are: 1) To characterize the structure-activity-function determinants for EPCR- dependent non-canonical PAR1 and PAR3 activation, and 2) To provide proof-of-concept for optimized engineered EPCR variants with improved characteristics. Successful completion of the proposed studies will increase our knowledge and understanding of vascular, thrombotic, and inflammatory diseases and may provide a platform for the development of novel therapeutic strategies for a variety of disorders in which thrombosis, apoptosis and inflammation contribute to pathogenesis.